The endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are the major signaling molecules for the cannabinoid receptors CB1 and CB2. All neurotransmitters require an efficient mechanism to terminate their action at the receptor. Water-soluble neurotransmitters are removed from the synapse by specific plasma transmembane transporters and they then diffuse unassisted within the cell to an enzyme for breakdown. The hypothesis of this proposal is that, owing to their neutral lipid structure the inactivation of AEA and 2-AG occurs by a unique pathway. It is proposed that this inactivation occurs by two steps: 1) Passive diffusion through the plasma membrane, independently of a transmembrane transporter, and 2) intracellular trafficking with chaperone proteins to their inactivating enzymes. To examine whether AEA and 2-AG can passively diffuse across the membrane, synthetic lipid vesicles (liposomes) will be employed with internalized FAAH (fatty acid amide hydrolase) or MAGL (monoacylglycerol lipase). These inactivating enzymes maintain the outward/inward gradient that drives the uptake. This model system will also be valuable in determining if simple diffusion demonstrates saturation kinetics, owing to an unstirred water layer, and if membrane composition regulates uptake. The latter will be addressed by comparing uptake in liposomes with and without lipid rafts. Of the possible mechanisms for the movement of AEA and 2-AG from the plasma membrane to their inactivating enzymes in the cell: unassisted simple diffusion, endocytosis, or trafficking by a chaperone protein, the latter appears most plausible. Accordingly, these proposed studies will examine endogenous levels of endocannabinoids trafficking proteins in cells and will elucidate the role of these proteins by comparing endocannabinoid metabolism rates and cellular uptake when the proteins are up-regulated by transfection and down-regulated by RNA interference or specific inhibitors. From a health related viewpoint, understanding the mechanism of endocannabinoid inactivation may lead to drug targets for addiction, mood disorders, pain and inflammation, cognition, and appetite regulation.